The Effects Of PFKP On The Biological Behavior And Aerobic Glycolysis Of Human Cervical Cancer Cells

Aims:Cervical cancer is a global public health problem,which seriously threatens women’s health.The incidence and mortality of cervical cancer remain high in many developing countries,and new treatments still need to be explored.Aerobic glycolysis,one of the characteristic features of tumor cells,produces lactic acid and ATP by consuming glucose.ATP and the metabolic intermediates glucose-6-phosphate and pyruvate promote cell synthesis,which in turn promotes tumor cell proliferation,while lactic acid can destroy extracellular matrix,promote the invasion and migration of tumor cells.In recent years,the platelet isoform of phosphofructokinase(PFKP)has been widely known as one of the key rate-limiting enzymes of tumor aerobic glycolysis.Some studies have shown that PFKP can cause changes in glucose metabolism and affect the expression of several genes related to glycolysis metabolism,suggesting that high expression of PFKP can promote glycolysis in tumor cells.The up-regulation of PFKP m RNA expression was found in neurocytoma,prostate cancer,liver cancer,breast cancer,kidney cancer and other solid tumors.However,the effects and mechanism of PFKP in cervical cancer remain unclear.The aim of this study was to investigate the effects of PFKP on biological behavior and aerobic glycolysis of human cervical cancer cells by gene transfection,and to provide experimental basis for the treatment of cervical cancer.Methods:(1)To detect the expression of PFKP in human cervical cancer and its impact on the prognosis of patients: using the data from Gene Expression Profiling Interactive Analysis(GEPIA)database,the expression levels of PFKP m RNA in cervical cancer and normal cervical tissues were analyzed;The Human Protein Atlas(HPA)database was used to analyze the protein expression of PFKP in cervical cancer and normal cervical tissues,and the Kaplan-Meier plotter database was used to analyze the correlation between PFKP and the prognosis of cervical cancer patients.(2)To investigate the effect of PFKP on the biological behavior of human cervical cancer cells: Western blot was used to detect the protein expression of PFKP in MS751,Hela,Siha and C-33 A cell lines.Then the cell lines with high expression of PFKP was silenced by siRNA,and the cell lines with low expression of PFKP were transfected with PFKP plasmid to overexpress PFKP gene.The effect of PFKP silencing or overexpression on the proliferation of human cervical cancer cells was detected by MTS,and the effect of PFKP silencing or overexpression on the apoptosis of human cervical cancer cells were detected by Annexin V-FITC/PI double staining.The effects of PFKP silencing or overexpression on the invasion and migration of human cervical cancer cells were detected by Transwell chamber assay and wound healing assay respectively.Western blot was used to detect the effect of PFKP silencing or overexpression on the expression of c-Myc,Sox2 and Nanog in human cervical cancer cells.(3)To investigate the effect of PFKP on aerobic glycolysis in human cervical cancer cells:the effects of PFKP silencing or overexpression on glucose uptake and lactate production of human cervical cancer cells were detected by glucose kit and lactate kit,respectively.Results:(1)The results of GEPIA and HPA showed that the expression of PFKP in cervical carcinoma tissues was significantly higher than in normal tissues.Kaplan-Meier plotter results showed that the survival rate of patients with high PFKP expression was significantly lower than patients that with low PFKP expression.(2)The expression of PFKP was the highest in MS751 cells,followed by He La cells,Si Ha cells and C-33 A cells.After silencing PFKP by siRNA,the protein level of PFKP in MS751 cells was significantly decreased(P < 0.05),and the protein level of PFKP in C-33 A cells and Si Ha cells were significantly increased after PFKP overexpression(P < 0.05).Compared with the control group,the proliferation of MS751 cells was significantly inhibited by PFKP silencing(P < 0.01),while the proliferation of C-33 A cells and Si Ha cells were significantly promoted by PFKP overexpression(P < 0.05).The results of Annexin V-FITC/PI double staining showed that silencing and overexpression of PFKP have no significant effect on apoptosis.The results of Transwell analysis suggested that the invasive ability of MS751 cells was significantly reduced by PFKP silencing(P < 0.05),the invasive ability of C-33 A cells and Si Ha cells were significantly enhanced by PFKP overexpression(P < 0.01).The wound healing experiment showed that the migration ability of MS751 cells could be inhibited by PFKP silencing(P < 0.01),the migration ability of C-33 A cells and Si Ha cells were significantly increased by PFKP overexpression(P < 0.05).Western blot results showed that by silencing PFKP gene,the protein expression of stem-related genes c-Myc,Sox2 and Nanog also were decreased significantly(P < 0.01),and after PFKP overexpression in C-33 A cells and Si Ha cells,compared with OE-NC of control group,the protein expression levels of c-Myc,Sox2 and Nanog were increased(P <0.05)(3)The results of lactate test and Glucose test showed that lactate production and glucose uptake in MS751 cells were significantly reduced by PFKP silencing,while lactate production and glucose uptake in C-33 A and Si Ha cells were increased significantly by PFKP overexpression(P < 0.01).Conclusion:(1)The expression level of PFKP m RNA and protein in human cervical cancer tissues was significantly higher than that in normal cervical tissues,and negatively correlated with the prognosis of patients with cervical cancer;(2)The proliferation,invasion and migration of human cervical cancer cells were significantly inhibited and the stemness of human cervical cancer cell was weaken by PFKP silencing;the proliferation,invasion and migration of human cervical cancer cells were significantly promoted and the stemness of human cervical cancer cell was enhanced by PFKP overexpression;(3)The aerobic glycolysis and glucose uptake and lactate production in human cervical cancer cells were inhibited by PFKP silencing,The aerobic glycolysis and increase glucose uptake and lactate production in human cervical cancer cells were promoted by PFKP overexpression.